Estimation of Spatiotemporal Gait Parameters in Walking on a Photoelectric System: Validation on Healthy Children by Standard Gait Analysis.

The use of stereophotogrammetry systems is challenging when targeting children's gait analysis due to the time required and the need to keep physical markers in place. For this reason, marker-less photoelectric systems appear to be a solution for accurate and fast gait analysis in youth. The aim of this study is to validate a photoelectric system and its configurations (LED filter setting) on healthy children, comparing the kinematic gait parameters with those obtained from a three-dimensional stereophotogrammetry system. Twenty-seven healthy children were enrolled. Three LED filter settings for the OptoGait were compared to the BTS P6000. The analysis included the non-parametric 80% limits of agreement and the intraclass correlation coefficient (ICC). Additionally, normalised limits of agreement and bias (NLoAs and Nbias) were compared to the clinical experience of physical therapists (i.e., assuming an error lower than 5% is acceptable). ICCs showed excellent consistency for most of the parameters and filter settings; NLoAs varied between 1.39% and 12.62%. An inverse association between the number of LEDs for filter setting and the bias values was also observed. Observations confirm the validity of the OptoGait system for the evaluation of spatiotemporal gait parameters in children.

Evaluation of sense of position and agency in children with diplegic cerebral palsy: A pilot study.

PURPOSE: This study explores non-motor impairment of the upper dominant limb in children with diplegic cerebral palsy (CP). Specifically, it firstly investigates sense of position without visual control, ability to compare visual and proprioceptive information, and capacity to recognize effects of self-movement. Secondly, it explores the possible association between these items with cognitive function, perceptual disorder (PD), and manual ability (Manual Ability Classification System -MACS). METHODS: Ten subjects (7 female; 3 male) were tested with three protocols: the first one (A) explored sense of position, the second one (B) collimation between visual and proprioceptive information, and the last one (C) sense of agency with the use of videogames. RESULTS: All subjects executed Protocol A without making any mistakes, while in Protocol B the median percentage of mistakes was 4.8%. Mistakes were recorded more frequently in positions characterized by wrist extension. Data reported a significant correlation with Processing Speed Index (PSI). Sense of agency was positively associated with cognitive functioning, with a significant correlation with PSI.PD, MACS, and Video-Gaming Experience (VGE) showed no correlation with Protocol C. CONCLUSION: In the observed sample with diplegic CP, preliminary data support the hypothesis that there is an alteration of both sense of position and sense of agency. They were both associated to PSI, with a positive trend of correlation with cognitive functioning. PD seemed to have no influence. Further studies, with a larger sample size, a control group, and involving children without CP, are required to corroborate the results obtained.

The AtMYB60 transcription factor regulates stomatal opening by modulating oxylipin synthesis in guard cells.

Stomata are epidermal pores formed by pairs of specialized guard cells, which regulate gas exchanges between the plant and the atmosphere. Modulation of transcription has emerged as an important level of regulation of stomatal activity. The AtMYB60 transcription factor was previously identified as a positive regulator of stomatal opening, although the details of its function remain unknown. Here, we propose a role for AtMYB60 as a negative modulator of oxylipins synthesis in stomata. The atmyb60-1 mutant shows reduced stomatal opening and accumulates increased levels of 12-oxo-phytodienoic acid (12-OPDA), jasmonic acid (JA) and jasmonoyl-L-isoleucine (JA-Ile) in guard cells. We provide evidence that 12-OPDA triggers stomatal closure independently of JA and cooperatively with abscisic acid (ABA) in atmyb60-1. Our study highlights the relevance of oxylipins metabolism in stomatal regulation and indicates AtMYB60 as transcriptional integrator of ABA and oxylipins responses in guard cells.

Quantitative and Qualitative Running Gait Analysis through an Innovative Video-Based Approach.

Quantitative and qualitative running gait analysis allows the early identification and the longitudinal monitoring of gait abnormalities linked to running-related injuries. A promising calibration- and marker-less video sensor-based technology (i.e., Graal), recently validated for walking gait, may also offer a time- and cost-efficient alternative to the gold-standard methods for running. This study aim was to ascertain the validity of an improved version of Graal for quantitative and qualitative analysis of running. In 33 healthy recreational runners (mean age 41 years), treadmill running at self-selected submaximal speed was simultaneously evaluated by a validated photosensor system (i.e., Optogait-the reference methodology) and by the video analysis of a posterior 30-fps video of the runner through the optimized version of Graal. Graal is video analysis software that provides a spectral analysis of the brightness over time for each pixel of the video, in order to identify its frequency contents. The two main frequencies of variation of the pixel's brightness (i.e., F1 and F2) correspond to the two most important frequencies of gait (i.e., stride frequency and cadence). The Optogait system recorded step length, cadence, and its variability (vCAD, a traditional index of gait quality). Graal provided a direct measurement of F2 (reflecting cadence), an indirect measure of step length, and two indexes of global gait quality (harmony and synchrony index). The correspondence between quantitative indexes (Cadence vs. F2 and step length vs. Graal step length) was tested via paired t-test, correlations, and Bland-Altman plots. The relationship between qualitative indexes (vCAD vs. Harmony and Synchrony Index) was investigated by correlation analysis. Cadence and step length were, respectively, not significantly different from and highly correlated with F2 (1.41 Hz ± 0.09 Hz vs. 1.42 Hz ± 0.08 Hz, p = 0.25, r2 = 0.81) and Graal step length (104.70 cm ± 013.27 cm vs. 107.56 cm ± 13.67 cm, p = 0.55, r2 = 0.98). Bland-Altman tests confirmed a non-significant bias and small imprecision between methods for both parameters. The vCAD was 1.84% ± 0.66%, and it was significantly correlated with neither the Harmony nor the Synchrony Index (0.21 ± 0.03, p = 0.92, r2 = 0.00038; 0.21 ± 0.96, p = 0.87, r2 = 0.00122). These findings confirm the validity of the optimized version of Graal for the measurement of quantitative indexes of gait. Hence, Graal constitutes an extremely time- and cost-efficient tool suitable for quantitative analysis of running. However, its validity for qualitative running gait analysis remains inconclusive and will require further evaluation in a wider range of absolute and relative running intensities in different individuals.

Testing the Performance of an Innovative Markerless Technique for Quantitative and Qualitative Gait Analysis.

Gait abnormalities such as high stride and step frequency/cadence (SF-stride/second, CAD-step/second), stride variability (SV) and low harmony may increase the risk of injuries and be a sentinel of medical conditions. This research aims to present a new markerless video-based technology for quantitative and qualitative gait analysis. 86 healthy individuals (mead age 32 years) performed a 90 s test on treadmill at self-selected walking speed. We measured SF and CAD by a photoelectric sensors system; then, we calculated average ± standard deviation (SD) and within-subject coefficient of variation (CV) of SF as an index of SV. We also recorded a 60 fps video of the patient. With a custom-designed web-based video analysis software, we performed a spectral analysis of the brightness over time for each pixel of the image, that reinstituted the frequency contents of the videos. The two main frequency contents (F1 and F2) from this analysis should reflect the forcing/dominant variables, i.e., SF and CAD. Then, a harmony index (HI) was calculated, that should reflect the proportion of the pixels of the image that move consistently with F1 or its supraharmonics. The higher the HI value, the less variable the gait. The correspondence SF-F1 and CAD-F2 was evaluated with both paired t-Test and correlation and the relationship between SV and HI with correlation. SF and CAD were not significantly different from and highly correlated with F1 (0.893 ± 0.080 Hz vs. 0.895 ± 0.084 Hz, p < 0.001, r2 = 0.99) and F2 (1.787 ± 0.163 Hz vs. 1.791 ± 0.165 Hz, p < 0.001, r2 = 0.97). The SV was 1.84% ± 0.66% and it was significantly and moderately correlated with HI (0.082 ± 0.028, p < 0.001, r2 = 0.13). The innovative video-based technique of global, markerless gait analysis proposed in our study accurately identifies the main frequency contents and the variability of gait in healthy individuals, thus providing a time-efficient, low-cost means to quantitatively and qualitatively study human locomotion.

Time-course of running treadmill adaptation in novice treadmill runners.

Studies on running biomechanics and energetics are usually conducted on a treadmill. To ensure that locomotion on a treadmill is comparable to locomotion overground, participants need to be expert in the use of the device. This study aimed to identify the number and duration of sessions needed to obtain stable measurements for spatiotemporal and metabolic parameters in unexperienced treadmill runners. Fourteen male recreational runners performed three 15-min treadmill running trials in different days at a submaximal speed. Spatiotemporal and metabolic parameters were registered at minutes: 5, 10, 15 and their within-trial and between-trial changes were analysed using a two-way repeated measures ANOVA and Bonferroni post-hoc test. Within-trial differences were found in step frequency (decreased over time), Step Length and Contact Time (increased), reaching stability at different time points. Ventilator parameters increased, reaching stability after 5-10 min, while heart rate increased progressively over time. The only between-trial differences were an increase in step length and a decrease in step frequency at min 1, between trials 1 and 3. In conclusion, at least three running trials of 15 min are required to familiarize with the device. The last 5 min of the third trial can be regarded as stable measurements.

Relationship between Lower Limb Kinematics and Upper Trunk Acceleration in Recreational Runners.

Upper trunk (UT) kinematics in runners and its relationship with lower limbs has been poorly investigated, although it is acknowledged that dynamic stability of the upper body is a primary objective of human locomotion. This study aimed to explore UT kinematics according to gender and level of training and in relation to lower limb run patterns described through the presence of: overstriding, crossover, excessive protonation, and pelvic drop. Lower body variables chosen to describe running pattern were those that are frequently modified during gait-retraining with the goal of reducing injury risk. Eighty-seven recreational runners (28 females and 59 males, age 41 ± 10 years) performed a one minute run test on a treadmill at self-selected speed. UT kinematics was measured using an inertial measurement unit, while run features were assessed through an optoelectronic system and video analysis. Accelerations and root-mean-square on mediolateral and anteroposterior axes, normalized using the vertical component of the acceleration, were estimated to describe UT stability. Results showed no significant differences in the normalized UT acceleration root-mean-square according to gender and level of training as well as according to the presence of overstriding, crossover, and excessive protonation. The only running strategy studied in this work that showed a significant relationship with UT stability was the presence of excessive pelvic drop. The latter was significantly associated (p=0.020) to a decrease in the normalized acceleration root-mean-square along the mediolateral direction. Although the excessive pelvic drop seemed to have a positive effect in stabilizing the upper body, concerns remain on the effect of a poor control of the pelvis on the biomechanics of lower limbs. Results obtained confirm the hypothesis that the lower body is able to respond to varying impact load conditions to maintain UT stability.

Abscisic Acid Is a Major Regulator of Grape Berry Ripening Onset: New Insights into ABA Signaling Network.

Grapevine is a world-wide cultivated economically relevant crop. The process of berry ripening is non-climacteric and does not rely on the sole ethylene signal. Abscisic acid (ABA) is recognized as an important hormone of ripening inception and color development in ripening berries. In order to elucidate the effect of this signal at the molecular level, pre-véraison berries were treated ex vivo for 20 h with 0.2 mM ABA and berry skin transcriptional modulation was studied by RNA-seq after the treatment and 24 h later, in the absence of exogenous ABA. This study highlighted that a small amount of ABA triggered its own biosynthesis and had a transcriptome-wide effect (1893 modulated genes) characterized by the amplification of the transcriptional response over time. By comparing this dataset with the many studies on ripening collected within the grapevine transcriptomic compendium Vespucci, an extended overlap between ABA- and ripening modulated gene sets was observed (71% of the genes), underpinning the role of this hormone in the regulation of berry ripening. The signaling network of ABA, encompassing ABA metabolism, transport and signaling cascade, has been analyzed in detail and expanded based on knowledge from other species in order to provide an integrated molecular description of this pathway at berry ripening onset. Expression data analysis was combined with in silico promoter analysis to identify candidate target genes of ABA responsive element binding protein 2 (VvABF2), a key upstream transcription factor of the ABA signaling cascade which is up-regulated at véraison and also by ABA treatments. Two transcription factors, VvMYB143 and VvNAC17, and two genes involved in protein degradation, Armadillo-like and Xerico-like genes, were selected for in vivo validation by VvABF2-mediated promoter trans-activation in tobacco. VvNAC17 and Armadillo-like promoters were induced by ABA via VvABF2, while VvMYB143 responded to ABA in a VvABF2-independent manner. This knowledge of the ABA cascade in berry skin contributes not only to the understanding of berry ripening regulation but might be useful to other areas of viticultural interest, such as bud dormancy regulation and drought stress tolerance.

Comparative analysis of fruit aroma patterns in the domesticated wild strawberries "Profumata di Tortona" (F. moschata) and "Regina delle Valli" (F. vesca).

Strawberry is one of the most valued fruit worldwide. Modern cultivated varieties (Fragaria × ananassa) exhibit large fruits, with intense color and prolonged shell life. Yet, these valuable traits were attained at the cost of the intensity and the variety of the aroma of the berry, two characteristics highly appreciated by consumers. Wild species display smaller fruits and reduced yield compared with cultivated varieties but they accumulate broader and augmented blends of volatile compounds. Because of the large diversity and strength of aromas occurring in natural and domesticated populations, plant breeders regard wild strawberries as important donors of novel scented molecules. Here we report a comprehensive metabolic map of the aroma of the wild strawberry Profumata di Tortona (PdT), an ancient clone of F. moschata, considered as one of the most fragrant strawberry types of all. Comparison with the more renowned woodland strawberry Regina delle Valli (RdV), an aromatic cultivar of F. vesca, revealed a significant enrichment in the total level of esters, alcohols and furanones and a reduction in the content of ketones in in the aroma of PdT berries. Among esters, particularly relevant was the enhanced accumulation of methyl anthranilate, responsible for the intensive sweetish impression of wild strawberries. Interestingly, increased ester accumulation in PdT fruits correlated with enhanced expression of the Strawberry Alcohol Acyltransferase (SAAT) gene, a key regulator of flavor biogenesis in ripening berries. We also detected a remarkable 900-fold increase in the level of mesifurane, the furanone conferring the typical caramel notes to most wild species.

The Arabidopsis thaliana MYB60 promoter provides a tool for the spatio-temporal control of gene expression in stomatal guard cells.

Plants have evolved different strategies to resist drought, of which the best understood is the abscisic acid (ABA)-induced closure of stomatal pores to reduce water loss by transpiration. The availability of useful promoters that allow for precise spatial and temporal control of gene expression in stomata is essential both for investigating stomatal regulation in model systems and for biotechnological applications in field crops. Previous work indicated that the regulatory region of the transcription factor AtMYB60 specifically drives gene expression in guard cells of Arabidopsis, although its activity is rapidly down-regulated by ABA. Here, the activity of the full-length and minimal AtMYB60 promoters is reported in rice (Oryza sativa), tobacco (Nicotiana tabacum), and tomato (Solanum lycopersicum), using a reporter gene approach. In rice, the activity of both promoters was completely abolished, whereas it was spatially restricted to guard cells in tobacco and tomato. To overcome the negative effect of ABA on the AtMYB60 promoter, a chimeric inducible system was developed, which combined the cellular specificity of the AtMYB60 minimal promoter with the positive responsiveness to dehydration and ABA of the rd29A promoter. Remarkably, the synthetic module specifically up-regulated gene expression in guard cells of Arabidopsis, tobacco, and tomato in response to dehydration or ABA. The comparative analysis of different native and synthetic regulatory modules derived from the AtMYB60 promoter offers new insights into the functional conservation of the cis-mechanisms that mediate gene expression in guard cells in distantly related dicotyledonous species and provides novel tools for modulating stomatal activity in plants.

Gene trap-based identification of a guard cell promoter in Arabidopsis.

Preserving crop yield under drought stress is a major challenge for modern agriculture. To cope with the detrimental effects of water scarcity on crop productivity it is important to develop new plants with a more sustainable use of water and capable of higher performance under stress conditions. Transpiration through stomatal pores accounts for over 90% of water loss in land plants. Recent studies have increased our understanding of the networks that control stomatal activity and have led to practical approaches for enhancing drought tolerance. Genetic engineering of target genes in stomata requires effective expression systems, including suitable promoters, because constitutive promoters (i.e., CaMV35S) are not always functional or can have negative effects on plant growth and productivity. Here we describe the identification of the CYP86A2 guard cell promoter and discuss its potential for gene expression in stomata.

Gene trap lines identify Arabidopsis genes expressed in stomatal guard cells.

We employed a gene trap approach to identify genes expressed in stomatal guard cells of Arabidopsis thaliana. We examined patterns of reporter gene expression in approximately 20,000 gene trap lines, and recovered five lines with exclusive or preferential expression in stomata. The screen yielded two insertions in annotated genes, encoding the CYTOCHROME P450 86A2 (CYP86A2) mono-oxygenase, and the PLEIOTROPIC DRUG RESISTANCE 3 (AtPDR3) transporter. Expression of the trapped genes in guard cells was confirmed by RT-PCR experiments in purified stomata. Examination of homozygous mutant lines revealed that abscisic acid (ABA)-induced stomatal closure was impaired in the atpdr3 mutant. In three lines, insertions occurred outside transcribed units. Expression analysis of the genes surrounding the trapping inserts identified two genes selectively expressed in guard cells, corresponding to a PP2C PROTEIN PHOSPHATASE and an unknown expressed protein gene. Statistical analyses of the chromosomal regions tagged by the gene trap insertions revealed an over-represented [A/T]AAAG motif, previously described as an essential cis-active element for gene expression in stomata. The lines described in this work identify novel genes involved in the modulation of stomatal activity, provide useful markers for the study of developmental pathways in guard cells, and are a valuable source of guard cell-specific promoters.